Method for the diagnosis of lymphoproliferative diseases

ABSTRACT

The present invention relates to novel methods for the diagnosis and therapy of lymphoproliferative diseases. Specifically, the present invention relates to novel methods for the diagnosis and therapy taking advantage of the detection of chromosomal breakpoints in chromosome 12 and/or translocation of chromosomal material from chromosome 12, said chromosomal breakpoints and/or translocation(s) being associated with lymphoproliferative diseases, such as primary cutaneous T-cell lymphomas (CTCL). The present invention further relates to the use of neuron navigator 3 gene (NAV3) or an equivalent or functional fragment thereof involved in chromosomal breakpoints in chromosome 12 and/or translocations thereof, said gene and/or translocations thereof being associated with lymphoproliferative diseases, such as primary cutaneous T-cell lymphomas (CTCL), as a diagnostic and therapeutic agent. The present invention also relates to the development of therapy.

This application is the US national phase of international applicationPCT/FI03/00061 filed 24 Jan. 2003, which designated the US and claimspriority to FI Application No. 20020132 filed 24 Jan. 2002 and FIApplication No. 20021617 filed 10 Sep. 2002. The entire contents ofthese applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel methods for the diagnosis andtherapy of lymphoproliferative diseases. Specifically, the presentinvention relates to novel methods for the diagnosis and therapy takingadvantage of the detection of chromosomal breakpoints in humanchromosome 12 and/or translocation of chromosomal material fromchromosome 12, said chromosomal breakpoints and/or translocation(s)being associated with lymphoproliferative diseases, such as primarycutaneous T-cell lymphomas (CTCL). The present invention further relatesto the use of neuron navigator 3 gene (NAV3) or an equivalent orfunctional fragment thereof involved in chromosomal breakpoints inchromosome 12 and/or translocations thereof, said gene and/ortranslocations thereof being associated with lymphoproliferativediseases, such as primary cutaneous T-cell lymphomas (CTCL), as adiagnostic and therapeutic agent. The present invention also relates tothe development of therapy.

BACKGROUND OF THE INVENTION

Primary cutaneous T-cell lymphomas (CTCL) represent a heterogeneousgroup of non-Hodgkin-lymphomas (NHL) whose etiology and pathomechanismare poorly understood [Siegel, R. S., et al., J. Clin. Oncol. 18 (2000)2908-2925]. There is no curative therapy for CTCL. During the last tenyears an increase of about 2-8% in the incidence of CTCL has beenobserved in the developed world [Doll, R., et al (Eds.), Trends incancer incidence and mortality, cancer surveys, Vol 19/20, Cold SpringHarbor Laboratory Press, 1994, 423-53; Hjalgrim, H., et al., Br. J.Cancer 73 (1996) 951-54]. After the group of primary gastrointestinallymphomas, CTCL together with the primary cutaneous B-cell lymphomasform the second most common group of extra-nodal NHL [Isaacson, P. G.and Norton, A. J., Cutaneous lymphoma, in Extra-nodal lymphomas, London,Churchill Livingstone, 1994, p. 172]. In Finland, a three-fold increasein the incidence of CTCL has been found in men, but not in women, duringthe last 40 years [Väkevä, L., et al., J. Invest. Dermatol. 115 (2000)62-65].

A major portion, about 80%, of the CTCL patients usually show anindolent disease course and long remissions may be achieved withtreatment. However, about 20% of the cases undergo a transformation intoan aggressive large-cell variant so that the tumour cells invade severaltissues, such as skin, blood, lymph nodes, and bone marrow, and there issome evidence that this is the original malignant cell clone [Wolfe, J.,et al., J. Clin. Oncol. 13 (1995) 1751-57]. The 5-year survival of thesepatients is below 15% [Willemze, R., et al., Blood 90 (1997) 354-371;Siegel, R. S., et al., supra]. This transformation cannot be predictedby any current means.

The most common form of CTCL is mycosis fungoides (MF). The first skinlesions develop slowly. They resemble eczema or mild psoriasis and arecalled Parapsoriasis en plaques (Pps). In the early phases, poly- oroligoclonal CD4-positive lymphocytes infiltrate towards the epidermis ofthe skin. The time and the compartment of the malignant transformationare not known [Veelken, H., et al., J. Invest. Dermatol. 104 (1995)889]. Malignant lymphocytes are later found also in blood and lymphnodes. However, recent data suggest that the malignant cells may bedispersed even earlier than previously thought [Karenko, L., et al., J.Invest. Dermatol. 108 (1997) 22-29; Karenko, L., et al., J. Invest.Dermatol. 112 (1999) 329-95: Karenko L, et al., J. Invest. Dermatol. 16(2001) 188-193].

A more aggressive form of CTCL is the leukaemic Sezary syndrome (SS),which may evolve from MF or begin directly with erythrodermic skinsymptoms.

In the treatment of CTCL, an early diagnosis is crucial, because thedisease is prone to relapse in later stages. However, no means areavailable at present for a definite diagnosis of CTCL. In particular, MFis difficult to diagnose in its early presentations due to itsresemblance to eczema or mild psoriasis and unfortunately may thusremain undetected in time. In the diagnosis of CTCL, a skin biopsysample is obtained from the affected skin area and a histopathologicalanalysis is performed. The histopathological diagnosis is based on thedetection of epidermotropic, morphologically malignant lymphocytes, i.e.cells with hyperchromatic, indented (cerebrifom) nuclei (Willemze, R.,et al., supra). In most, but not all, cases these cells express the CD4surface marker, which may be detected immunohistochemically (Willemze,R., et al., supra). Thus, the diagnostic accuracy depends on visualgrading and impression made by a pathologist, and early lesions withonly very few malignant cells or with chromosomally clonal malignantlymphocytes with as yet normal morphology may be missed.

Additionally, the demonstration of T-cell receptor (TCR) rearrangementin blood or skin lesion-derived DNA has been used as a supplementarymethod in the diagnosis of CTCL. However, TCR rearrangement is not adisease-specific marker, since it identifies also reactive clonal cells[Guitart J. and Kaul K., Arch. Dermatol. 135 (1999) 158-62].

Also several chromosomal aberrations, especially numerical aberrations,have been observed in CTCL in molecular cytogenetic studies and thesecan be used in the diagnosis. In particular, the cytogenetic changeshave been shown to precede the histologically identifiable malignancy[Whang-Peng, J., et al., Cancer 50 (1982) 1539; Berger, R., et al.,Cancer Genet Cytogenet 27 (1987) 79; Karenko et al., 1997, supra].However, in the early phases of the disease these abnormalities havebeen non-clonal, which renders the detection non-specific.

It is of highest importance to develop new methods, which enable anearly diagnosis and the follow-up of therapeutic interventions in termsof residual malignant cells in lymphoproliferative diseases, such asCTCL. Also, additional means for the development of new guidelines forthe initiation and follow-up of therapy are greatly needed.

SUMMARY OF THE INVENTION

We have now identified specific recurrent chromosomal breakpoints and/ortranslocations, which are associated with cutaneous T-cell lymphoma(CTCL), in chromosome 12, specifically in 12q14-12q24. We have alsoidentified a gene involved in such chromosomal breakpoints and/ortranslocations. The identification of such specific chromosomalbreakpoints and/or translocations and the gene and/or translocation,deletions or other defects of the gene allows the development of noveldiagnostic and therapeutic methods for the detection and follow-up andtreatment of lymphoproliferative diseases, such as CTCL.

The object of the invention is to provide novel methods and means forthe diagnosis of lymphoproliferative diseases, such as CTCL, suchmethods and means allowing an early diagnosis of the disease.

Another object of the invention is to provide novel methods and meansfor the diagnosis of lymphoproliferative diseases, such as CTCL, suchmethods and means being specific and reliable.

Yet another object of the invention is to provide novel methods andmeans for the prediction of the progression of the disease and itstransformation to an aggressive form in lymphoproliferative diseases,such as CTLC, such methods and means allowing a timely therapeuticintervention, which may be life-saving.

Still another object of the invention is to provide novel methods andmeans for the development of new guidelines for the initiation andfollow-up of therapeutic interventions as well as for the development ofnew treatment modalities for lymphoproliferative diseases, such as CTCL,such methods and means prolonging the remission stage of the disease andintroducing new possibilities for combating the disease and for therecovery of the patient.

The present invention relates to a novel method for the diagnosis andthe follow-up of the lymphoproliferative diseases, such as CTCL,comprising the detection of the presence or the absence of at least onespecific chromosomal breakpoint in chromosome 12 and/or at least onetranslocation of chromosomal material from chromosome 12, saidchromosomal breakpoint and/or translocation being associated with saidlymphoproliferative diseases, in a clinical sample.

The present invention further relates to a novel method for thediagnosis and the follow-up of the lymphoproliferative diseases, such asCTCL, comprising the detection of the presence or the absence of neuronnavigator 3 (NAV3) gene or an equivalent or a functional fragmentthereof associated with said lymphoproliferative diseases in a clinicalsample.

The present invention still further relates to a novel method for thediagnosis and the follow-up of the lymphoproliferative diseases, such asCTCL, comprising the detection of the presence or the absence of atranslocation or a deletion or another defect of neuron navigator 3(NAV3) or an equivalent or fragment thereof in chromosome 12 associatedwith said lymphoproliferative diseases in a clinical sample.

The present invention also relates to rapid test systems for theidentification of molecular cytogenetic alterations in clinicalspecimens obtained from patients suffering from a lymphoproliferativedisease, such as CTCL, based on the detection of at least one specificchromosomal breakpoint in chromosome 12 and/or at least onetranslocation of chromosomal material from chromosome 12 and/or atranslocation or a deletion or another defect of neuron navigator 3(NAV3) gene or an equivalent or a functional fragment thereof inchromosome 12, said chromosomal breakpoint and/or translocation and/orgene translocation, deletion or defect being associated with thecytogenetic alterations.

The present invention further relates to a method of identifyingpatients who are in the risk of developing a lymphoproliferativedisease, such as CTCL, or its leukaemic variant, and who could be helpedby a timely therapeutic interaction by detecting the presence or absenceof at least one specific chromosomal breakpoint in chromosome 12 and/orat least one translocation of chromosomal material from chromosome 12and/or a translocation or a deletion or another defect of neuronnavigator 3 (NAV3) gene or an equivalent or functional fragment thereofin chromosome 12, said chromosomal breakpoint and/or translocationand/or gene translocation, deletion or defect being associated with thedisease subtypes in a biological sample obtained from said patients.

The present invention further relates to a method of predicting theprogression of lymphoproliferative diseases, such as CTLC, and thetransformation thereof to an aggressive variant by detecting thepresence or absence of at least one specific chromosomal breakpoint inchromosome 12, specifically in 12q14-12q24, and/or at least onetranslocation of chromosomal material from chromosome 12 and/or atranslocation or a deletion or another defect of neuron navigator 3(NAV3) gene or an equivalent or a fragment thereof in chromosome 12,said chromosomal breakpoint and/or translocation and/or genetranslocation, deletion or defect being associated with the diseasesubtypes, in a biological sample obtained from a patient suffering fromsaid disease.

The present invention also relates to a use of a specific chromosomalbreakpoint or specific chromosomal breakpoints in chromosome 12,specifically in 12q14-12q24, and/or a translocation of chromosomalmaterial from chromosome 12, said chromosomal breakpoint and/ortranslocation being associated with lymphoproliferative diseases, suchas CTCL, for the diagnosis of said diseases.

The present invention also relates to a use of neuron navigator 3 (NAV3)gene or an equivalent or a fragment thereof and/or of a translocation, adeletion or another defect of neuron navigator 3 (NAV3) gene or anequivalent or a fragment thereof in chromosome 12, said gene,translocation, deletion or defect being associated withlymphoproliferative diseases, such as CTCL, for the diagnosis of saiddiseases.

The present invention also relates to a use of a specific chromosomalbreakpoint or chromosomal breakpoints in chromosome 12 and/or atranslocation of chromosomal material from chromosome 12, saidchromosomal breakpoint and translocation being associated withlymphoproliferative diseases, such as CTCL, for the development oftherapy of said diseases.

The present invention also relates to a use of neuron navigator 3 (NAV3)gene or an equivalent or a fragment thereof and/or of a translocation,deletion or another defect of neuron navigator 3 (NAV3) gene anequivalent or a fragment thereof in chromosome 12 associated withlymphoproliferative diseases, such as CTCL, in therapy of said diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a SKY analysis of a translocation between chromosomes 12qand chromosome 18q in a sample from a SS patient. Normal chromosomes aremarked with N, translocated chromosomes with T.

FIG. 2 shows a FISH-analysis of translocations between chromosomes 12q,4q and 10. The uppermost sub-window shows a normal chromosome 4. Thesecond sub-window shows a chromosome 4 with translocated material ofchromosome 12 distally in the q-arm. The third sub-window showschromosome 12q with a breakpoint in q22, and translocated material ofchromosome 10 distally in the q-arm. The lowest sub-window showschromosome 10. The combined colours of FISH of each chromosome are shownon the right-hand side of each sub-window (N=normal, T=translocation).

FIG. 3 shows a FISH analysis of a chromosome sample. FIGS. 3A) and 3B)represent signal combinations of chromosomes 12 and 18 in the same cell.Combined signals of the chromosomes are seen on the right-hand side inboth pictures. The signal spectra are seen in the middle sub-windows.The sample was obtained from the same patient as in FIG. 1.

FIG. 3A). The uppermost sub-window shows a normal chromosome 12 (N1),which is also the middle chromosome in the group of three chromosomes 12on the right-hand side of the background picture. The centromere ofchromosome 12 is visible. YAC 803-C-2 is indicated in sub-windows and inthe background pictures. BAC RP11-359M6 is visible in the uppermostsub-window and in the chromosome 12 in the middle in the group ofchromosomes 12. The signal is missing in the two next sub-windowsshowing that it has moved away from the two other copies (T1 and T2) ofchromosome 12. The signal is lacking also from the chromosomes on bothsides of the normal chromosome 12 in the background picture, whereas theYAC 803-C-2 signal indicating presence of YAC 803-C-2 derived materialis present in these two copies. The lowest sub-window depicts chromosome18 (T3) that has received material corresponding to BAC RP11-359M6. Thecentromere of chromosome 18 is the upper signal and the lower signal isBAC. The corresponding chromosome 18 showing the translocated materialis the leftmost (T3) chromosome 18 in the group of four chromosomes 18in the background picture.

FIG. 3B). Two upper sub-windows represent normal chromosomes 18 (N2 andN3) showing only centromeres. The third sub-window shows chromosome 18(T4) that has received material from chromosome 12 (the BAC 359M6 is theother signal under the centromere as indicated in FIG. 3B). Thischromosome (T4) is the second from right in the background picture.

FIG. 4 shows a FISH-analysis of a translocation between chromosomes 12and 18 using BAC 36P3 and YAC 857F6. The uppermost sub-window shows anormal chromosome 12: the centromere signal; a small BAC 36P3 signalunder the centromere; and YAC 857F6 signal are indicated in FIG. 4. Thesecond sub-window shows aberrant chromosome 12, from which chromosomalmaterial has translocated to chromosome 18. The centromere signal isindicated in FIG. 4; BAC 36P3 is no more detected; YAC 857F6 isthe-green signal indicated in FIG. 4. The third sub-window shows anaberrant chromosome 18: the centromere gives a signal as indicated inFIG. 4; BAC36P3 gives the signal indicating translocated material fromchromosome 12. The lowest sub-window shows normal chromosome 18: thecentromere gives the signal as indicated in the FIG. 4. The combinedsignals of FISH of each chromosome are shown on the right hand side ofeach sub-window. The upper background picture shows normal and aberrantchromosome 12 on the left hand and right hand side, respectively, andthe lower background picture shows normal and aberrant chromosome 18 onthe right-hand and left-hand side, respectively.

FIG. 5 shows a FISH-analysis of a translocation between chromosomes 12(centromere signal indicated in FIG. 5) and 18q (centromere signalindicated in FIG. 5) with BAC 36P3 in a sample from a SS patient. Theupper row from the left to the right: two aberrant chromosomes 12 andone normal chromosome 12; the centromere of chromosome 12 and BAC36P3both give signals as indicated in FIG. 5, which are merged due to theshortness of chromosome 12. The lower row from left to the right: twoaberrant chromosomes 18 and two normal chromosomes 18 with thecentromere giving the signal as indicated in FIG. 5, and thetranslocated BAC 36P3 the signal indicated in FIG. 5.

FIG. 6 shows a FISH-analysis of translocations between chromosomes 12and 18 using BAC 781A6 (signal indicated in FIG. 6) and BAC 136F16(signals indicated in FIG. 6). The uppermost sub-window shows chromosome12 after translocation: the centromere signal is indicated in FIG. 6;BAC 136F16 is absent; BAC 781A6 gives a signal indicated in FIG. 6. Thesecond sub-window shows a normal chromosome 12: the centromere signal isindicated in FIG. 6; BAC 136F16 gives the double signal indicated inFIG. 6 under the centromere; BAC 781A6 gives the signal indicated inFIG. 6. The third sub-window shows normal chromosome 18 without specificsignals. The lowest sub-window shows chromosome 18 after translocation:BAC 136F16 gives double signal indicated in FIG. 6. The combined signalsof FISH of each chromosome are shown on the right-hand side of eachsub-window. The upper background picture shows normal and aberrantchromosome 12 on the right hand and left hand side, respectively, andthe lower background picture shows normal and aberrant chromosome 18 onthe left-hand and right-hand side, respectively.

FIG. 7 shows a FISH-analysis of chromosome 12 using BAC494K17 (signalindicated in FIG. 7) and BAC 144J4 (12q24)(signal indicated in FIG. 7).The upper sub-window shows normal chromosome 12: the centromere gives asignal indicated in FIG. 7. The lower sub-window shows chromosome 12after translocation: BAC 494K17 (signal indicated in FIG. 7) remains;BAC 144J4 has disappeared.

FIG. 8 shows the signals of YAC, BAC or Pac (P1) probes in a FISH assaywith some corresponding genes in aberrant chromosome 12 observed inpatients 1, 2 and 3 all suffering from SS, the leukaemic subtype ofCTCL, demonstrating the long chromosomal distances studied withhybridisations

FIG. 9 shows the parts of the NAV3 gene involved in translocation inpatient 3 as detected with FISH performed using BAC probes.

The exact breakpoint (question mark) within the limits of BACRP11-494K17 is an approximation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of a specificbreakpoint or specific breakpoints in chromosome 12q, specificallybetween 12q14-q24, with or without translocation of chromosomal materialto other chromosomes, which breakpoints are associated with CTCL.

As used herein the terms “chromosomal breakpoint” and “chromosomalbreak” refer to a point from which the chromosome has lost material andis thus a structural abnormality of the chromosome. As used herein, theterm “translocation” refers to transfer of chromosomal regions betweennon-homologous chromosomes.

As used herein the term “neuron navigator 3 gene” (NAV3) refers to thegene having the sequence identified as Seq. Id. Nr. 1 or an equivalentthereof having essentially the same function and encoding essentiallythe same protein, or that having the sequence identified as Seq. Id. Nr.3 or an equivalent thereof. The term “equivalent” further includesfunctional fragments or variants of NAV3, such as the sequenceidentified as Seq. Id. 2, or a functionally equivalent isolated DNAsequences hybridizable thereto.

As used herein the term “functionally equivalent fragments” of NAV3 generefer to such gene fragments, which are detectable in the methods of theinvention.

As used herein the expression “deletion or another defect” of NAV3 generefers to the absence of a nucleotide or nucleotides and/or an exon orexons in the gene sequence which absence adversely affects the functionof the gene.

A breakpoint was observed in the region 12q14-12q24 in 6 of 7 patientshaving Sezary syndrome (SS), the leukaemic form of CTCL, and in 3 of 4patients with the Mycosis fungoides (MF; stage IA-IIB) subtype of CTCL(see FIGS. 1 and 2). In two patients, the breakpoint was specified tothe region 12q14-q21.3 (FIG. 3). In one of these latter patients, thedeletion in 12q was interstitial, so that the distal breakpoint was in12q23 or 12q24. In 3 patients with SS the chromosome material wastranslocated to chromosome 18p or 18q12-18q21. In one SS patienttranslocation of chromosome 12 derived material occurred in chromosome 4(FIG. 2) and a translocation to chromosome 22 was also observed.

The chromosomal breakpoints and translocations were identified withmulticolour fluorescent in situ-hybridisation: either with combinatorialmulti-fluor FISH [MFISH, Speicher, M. R., et al., Nat. Genet. 2 (1996)368-375] or with spectral karyotyping [SKY; Schröck, E., et al., Science273 (1966) 494-497]. The translocations and breakpoints were furtherspecified with locus-specific commercial probes, YAC 803-C-2 (12q14-q15)and BAC RP11-359M6, with FISH. The Yac 803-C-2 is part of a publishedYAC-contig of 12q15 [Scoenmakers, et al., Genomics 29 (1995) 665-678].It is located between DNA-markers STS 12-98 and STS-72, including thosemarkers, and is situated near the proximal end of the contig. TheYac803-C-2 is not chimeric. The YAC 803-C-2 contains high mobility groupprotein gene HMGI-C [Schoenmakers, et al., Nature Genetics 10 (1995)436-444; Schoenmakers, et al., Genomics 29 (1995) 665-678]. The BACRP11-359M6 is published [AC027288.26 8422 . . . 8574 in the www dot ncbidot nlm dot nih dot gov (Roswell Park Cancer Institute Human BACLibrary, complete sequence 177080]. The BAC RP11-359M6 contains thehuman PAWR-gene [localization 12q21; Johnstone, et al., Genomics 53(1998) 241-243; for BAC, see www dot ncbi dot nlm dot nih dot gov]. The12q24 is known to contain a retropseudogene HMGIY [Rogalla, et al.,Cancer Genet. Cytogenet. 130 (2001) 51-56], which is another member ofthe high mobility group protein genes. In uterine leiomyomas, themitochondrial aldehyde dehydrogenase (ALDH2) gene in 12q24.1 has beenfound a translocation partner to HMGIC [Kazmierczak, et al., Cancer Res55 (1995) 6038-6039].

By a FISH analysis, the chromosomal breakpoint in 12q14-q21 and itstranslocation to 18q12-q21 in a biological sample obtained from an SSpatient were observed as a disappearance of the green BAC RP11-359M6spot from chromosome 12 (FIG. 3A, the uppermost sub-window, the normalchromosome, versus the two next sub-windows, defective chromosomes) andas an appearance of an additional green BAC RP11-359M6 spot inchromosome 18 (FIG. 3B, the two upper sub-windows, normal chromosomes,versus the lowest sub-window, the defective chromosome 18).

As a consequence of the chromosomal break and translocation, a tumoursuppressor gene may be disrupted, or the transcription of a genepromoting cell growth or apoptosis may be amplified, or a neogeneintervening with known transcription factors regulating cell growth maybe formed inducing the transformation of the disease to its malignantform.

In order to further identify the breakpoints and translocations observedas well as to identify the translocated material further analyses withadditional specific YAC- and BAC-probes were performed (FIG. 8).

Probe YAC 855F7 was seen to be divided between the aberrant chromosome12 and the aberrant chromosome 18 showing that the translocationbreakpoint in a patient sample studied was within the limits of thisYAC. YAC 855F7 is part of the YAC-contig 12.4 (NIH: www dot ncbi dot nlmdot nih dot gov) and spans the region between markers CHLC.GATA65A12 andWI-6487. NCBI database revealed between those markers in thecorresponding genomic contig (NT_(—)009551) consecutive loci, i.e.,LOC255379, LOC255315, LOC204040, LOC121318, and KIAA0938. According toPCR with locus-specific STS-markers and a BLASTA computer analysis (wwwdot ncbi dot nlm dot nih dot gov), these loci were found to be situatedin YAC 855F7 DNA and four BACs: RP11-781A6, RP11-494K17, RP11-136F16,and RP11-36P3 (accession numbers AC073552.1, AC022268.5, AC073571.14,and AC073608.19, respectively).

The BAC RP11-494K17 contains locus 255315 and almost the whole locus204040. A part of the latter locus is also present in BAC 136F16, whichalso contains the whole locus 121318 and a small part of KIAA0938. TheBAC RP11-36P3 contains the whole locus KIAA0938, but not the sequence oflocus 121318. The BAC 781A6 contains locus 255379 and a small part oflocus 255315 (see FIG. 8)

According to the BLASTA-analysis and Maes et al. [Genomics 80 (2002)21-30], loci 255315, 203040, 121318, and KIAA0938 together form aRAINB1-gene homologue, neuron navigator 3 (NAV3) gene (Seq. Id. Nr. 1)or a partial coding sequence thereof (AF397731). LOC 255315 (Seq. Id.Nr. 2) shares its sequences 498 to 668 with the NAV3 sequence 1 to 171except for one nucleotide in position 594. A NAV3 gene sequencecontaining full 5′ terminal is set forth as Seq. Id. Nr.3.

By a FISH analysis, the above-observed chromosomal breakpoint in12q14-q21 and the translocation t(12;18)(q21;q12-21) in a biologicalsample obtained from an SS patient were confirmed by the disappearanceof the red BAC 36P3 spot from chromosome 12 (FIG. 4A, the uppermostsub-window, the normal chromosome, versus the next sub-window, theaberrant chromosome) and by the appearance of an additional red BAC 36P3spot in chromosome 18 (FIG. 4, the third and fourth sub-windows,aberrant chromosome 18 and normal chromosome 18, respectively). YAC857F6 remained in chromosome 12. The translocation of chromosomalmaterial from chromosome 12 to chromosome 18 was observed also by aSKY-analysis (FIG. 1).

A translocation between chromosomes 12 (centromere red) and 18q(centromere green) with BAC 36P3 in a sample from a SS patient wasfurther confirmed by an additional FISH-analysis (FIG. 5)

In a further FISH analysis with a SS patient sample, BAC 781A6 (a greensignal in FIG. 6) and BAC136F16 (a red and a wine red signal in FIG. 6)separate from each other in the translocation as judged from the absenceof the red spot and the presence of green spot in the aberrantchromosome 12 when compared to normal chromosome 12. This material hastranslocated to chromosome 18 (see red spot in the aberrant chromosome18 in FIG. 6). On the other hand, BAC 494K17 remains in chromosome 12(the green signal in aberrant chromosome 12 in FIG. 7) and BAC 144J4moves away from chromosome 12 (a missing red signal in aberrantchromosome 12 in FIG. 7)

As the BACs above contain loci that together form the NAV3 gene, theseparation of signals indicating the separation of loci shows thesplitting up of the NAV3 gene to two parts in the translocation, onepart remaining in chromosome 12 (the parts and loci situated in BACsRP11-786A1 and 494K17), the other part (the parts and loci situated inBACs 136F16 and 36P3) translocating to chromosome 18q.

The breakpoint deduced from by the FISH-experiments is situated in thedistal part of the region covered by BAC 494K17 (possibly locus 204040,FIG. 8). More exact definition will require DNA-level studies, forexample, cloning of the translocation breakpoint.

This is the first time that a specific recurrent breakpoint ortranslocation has been observed in CTCL, although other malignancies ofthe hematopoietic system with chromosomal break points/translocationshave been described. The best known is the translocation betweenchromosomes 9 and 22, resulting in the so-called Philadelphiachromosome, which has been identified in CML [Nowell, P. and Hungerford,D., Science 132 (1960) 1497; Rowley, J., Nature 243 (1973) 290-293.].This transformation results in the fusion of BCR and ABL genes, which isessential for the development of CML [Shtivelman, E., et al., Nature 315(1985) 550-554]. The fusion gene has tyrosine kinase activity. The MLL(Mixed Lineage Leukemia) gene is a common target for chromosomaltranslocations associated with human acute leukemias. The lattertranslocations result in a gain of MLL function by generating novelchimeric proteins containing the amino-terminus of MLL fused in-framewith one of 30 distinct partner proteins. Expression of this fusionprotein is necessary but not sufficient for leukemogenesis in the mousemodel [Ayton, P. and Cleary, M., Oncogene 20 (2001) 5695-707].

Acute promyelocytic leukemia is characterized by t(15;17) translocation(Xu et al., Leukemia 9 (2001) 1358-68], splenic marginal zone B-celllymphoma by t(11:14)(p11;q32) translocation [Cuneo, A., et al., Leukemia15 (2001) 1262-7], follicular lymphomas have t(14;18)(q32;q21) whichresults in the juxtaposition of the promoter region of IGH gene with thecoding region of the anti-apoptotic protein Bcl-2 on chromosome 18[Fukuhara, S., et al., Cancer Res 39 (1979) 3119-3128, Tsujimoto, Y. etal., Science 266 (1984) 1097-1099]. A recurrent, reciprocal balancedtranslocation t(2;5)(p23;q35) has been found in CD30+ anaplasticlarge-cell lymphomas [Kadin, M. E. and Morris, S. W., Leuk Lymphoma 29(1998) 249-56] and in up to 50% of CD30+ primary CTCL [Beylot-Barry, M.et al., Blood 91 (1998) 4668-76]. This translocation creates a novelfusion protein, which has transforming properties in vitro.

Similarly, this is the first time that a specific gene, NAV3, andtranslocations thereof have been associated with lymphoproliferativediseases, such as CTCL.

Neuron navigator 3 (NAV3) gene is a member of a recently identifiedhuman gene family, which shows homology to the unc-53, a cell guidancegene from Caenorhabditis elegans (Maes et al., supra). It also shareshomologous sequences with human RAINB1 (retinoic acid inducible inneuroblastoma cells) a mammalian homologue of unc-53 [Merrill et al.,PNAS 99 (2002) 3422-3427]. NAV3 consists of 39 exons and its expression,based on mRNA detection, is largely restricted to the brain tissue (Maeset al., 2002, supra), but no haematopoietic or lymphoid tissues havebeen examined until now. NAV3 was shown to produce transcripts encodingproteins of different lengths and it may be subject to tissue-specificalternative splicing. The sub-cellular localization of NAV3 is notknown. The homologous protein UNC-53, based on predicted structure, hastwo polyproline-rich domains that may represent SH3-binding domains, adoublet of central coiled-coil regions (possibly interacting with otherproteins) and a putative ATP/GTP-nucleotide binding site. It alsocontains two putative actin-binding domains [Stringham, E., et al.,Development 129 (2002) 3367-3379).

Based on the current knowledge of the aforementioned homologous genesand proteins, we assume that NAV3, as set forth in Seq. Id. Nr. 1 or Nr.3, exerts its function in signal transduction, and its absence (genedeletion) or disturbed or amplified function (as a consequence oftranslocation) provides a growth favour, possibly via regulatingresponse to apoptosis, for the cells, which in turn is the prerequisitefor malignancy.

Retinoids are known to exert therapeutic activity in CTCL [Zackheim, H.S., Dermatology 199 (1999) 102-105), and since the NAV3 gene is likelyto be induced by all-trans retinoic acid like its aforementionedhomologues, it is understandable that the deletion/translocation of NAV3as shown herein provides resistance to such therapy in vivo (as was thecase with all three patients studied in the experiments shown herein).

Additionally, the present invention identifies a new potential functionfor NAV3 gene by showing its involvement in a lymphoproliferativedisease, namely CTCL and especially, its leukaemic form Sezary syndrome.

The present invention characterizes cytogenetic findings and identifiesthe T cell clones with specific cytogenetic aberrations. The identifiedcells are truly malignant by definition, and the demonstration of suchcells in clinical tissue specimen indicates the presence of the disease.

The present invention identifies translocations and eventual neogeneformation as well as specific gene deletions and translocationsassociated with a lymphoproliferative disease, namely CTCL andespecially, its leukaemic form Sezary syndrome. By integrating the aboveknowledge, a pathophysiological scheme with model characters for otherlymphoproliferative diseases, new diagnostic tools are provided.

According to the diagnostic method of the present invention, thepresence or absence of a chromosomal breakpoint or breakpoints can bedetected from a biological sample by any known detection method suitablefor detecting the breakpoints and translocations. Such methods areeasily recognized by those skilled in the art and include fluorescencein situ hybridisations, such as multi-colour fluorescence in situhybridisations, that are based on chromosome-specific or arm-specificpainting probes that paint chromosome 12 with a specific colour,spectrum, colour ratio or colour intensity or their combination. Thepainting probe can be used alone or combined with other painting probesdetecting other chromosomes or chromosome arms as in multi-fluor insitu-hybridisation [MFISH, described by Speicher, M. R., et al., NatGenet 12 (1996) 368-375], or in spectral karyotyping [SKY, described bySchröck, E., et al, Science 273 (1996) 494-497], or in Combined binaryratio labelling [COBRA, described by Tanke, H. J., et al., Eur J. Hum.Genet 7 (1999) 2-11] or in colour changing karyotyping [CCK, describedby Henegariu, O., et al., Nat. Genet. 23 (1999) 263-4], or withcentromere specific probes, or with locus-specific or band-specificprobes for loci in the translocating regions. Even the conventionalG-banding techniques can be used in cases were the coarse detection ofthe translocation is regarded as sufficient. Preferable methods arethose suitable for use in clinical laboratories, such as MFISH and SKY.

According to one preferred embodiment of the present invention, whichtakes advantage of the identification of NAV3 gene in thelymphoproliferative diseases, the presence or absence of the NAV3 geneor an equivalent or a fragment thereof can be detected from a biologicalsample by any known detection method suitable for detecting a geneexpression (or copy number), i.e. methods based on detecting the copynumber of the gene (or DNA) and/or those based on detecting the geneexpression products (mRNA or protein). Such methods are easilyrecognized by those skilled in the art and include in situhybridisations, such as fluorescence in situ hybridisation (FISH), mRNAin situ hybridisation, Northern analysis, RT-PCR, Southern and Westernanalyses, immunohistochemistry, and other immunoassays, such as ELISA.Preferable methods are those suitable for use in routine clinicallaboratories, such as FISH, RT-PCR methods and immunohistochemistry.

In therapy, restoration of the normal function of the NAV3 gene can beused. This may be reached by enhancing the expression of functionallyhomologous genes, by introducing an intact NAV3 gene or by using analtered form of the NAV3 gene or antisense oligonucleotide against theNAV3 in any technique presently available for gene therapy to preventthe progression of a proliferating disease. In particular, tumor cellgrowth may be slowed down or even stopped by such therapy. Suchtechniques include the ex vivo and in situ therapy methods, the formercomprising transducing or transfecting an intact or altered NAV3 gene(or its functional domains) in a recombinant or peptide form or asantisense oligonucleotides or in a vector to the patient, and the lattercomprising inserting the altered gene or oligonucleotide into a carrier,which is then introduced into the patient. Depending on the disease tobe treated, a transient cure or a permanent cure may be achieved.Alternatively, monoclonal or humanized antibodies or peptides binding tothe NAV3 protein or to the fusion gene generated as a result of thetranslocation, can be used to suppress the function of the altered NAV3protein and thus tumor cell growth may be slowed down or even stopped.Antibodies against NAV3 could also be used to carry other agents, suchas cytotoxic substances, to the cancer cells over-expressing the NAV3gene. Such agents could then be used to kill specifically the cancercells.

The present invention also allows the development of rapid test systemsfor the identified molecular cytogenetic alterations in clinicalspecimens. Such systems include two DNA probes, which span thechromosomal breakpoint(s) and are labelled with different colourigenicor fluorescent markers, and which hybridise to easily obtainablenon-dividing skin or blood cells (interphase cells). In case thechromosomal breakpoint in the region between the probes is present inthe sample, the visualized signals either visually depart from eachother, disappear or merge. Probes useful in this embodiment of theinvention are, for example, those based on YAC:s or BAC:s, P1s orcosmids containing human locus-specific sequences. See also Example 2.The probes may be further developed by different procedures, for examplePCR for enrichment of the human insert of the YAC, and differentlabelling procedures.

One embodiment of the invention is a diagnostic kit, which comprisesreagents necessary for the detection of NAV3 gene, gene products orfragments thereof. These reagents can include specific antibodies,preferably monoclonal antibodies, capable of identifying NAV3 or itsgene products or fragments thereof, other antibodies, markers andstandards that are needed for visualization or quantification as well asbuffers, diluents, washing solutions and the like, commonly contained ina commercial reagent kit. Alternatively, the diagnostic kit of thepresent invention may comprise NAV3 gene product or its functionalvariant or fragment together with suitable reagents, such as thoselisted above, needed for the detection of the antibodies against theNAV3 protein.

In the method of the invention, the biological sample can be anysuitable tissue sample, such as a biopsy from the skin or lymph node, abody fluid, such as whole blood, lymph, or cerebrospinal fluid sample.The biological sample can be, if necessary, pretreated in a suitablemanner known to those skilled in the art.

The detection of chromosomal breakpoints in chromosome 12 and/ortranslocation thereof is valuable especially in early diagnosis and inpredicting the progression/transformation of lymphoproliferativediseases, such as primary cutaneous T-cell lymphomas (CTCL), by acombined diagnostic approach. For instance in CTCL, the recurrentbreakpoint(s) in chromosome 12 and/or translocation(s) thereof detectedin accordance with the present invention for the first time identify theaggressive forms of CTCL and, importantly, at an early stage of thedisease. However, combining the demonstration of this chromosomaltranslocation with those to be described later on will provide a basisfor creation of a pattern of chromosomal aberrations associated with aspecific clinical course of lymphoproliferative diseases. Also, toconfirm the functional capacity of the lymphocytes affected, theinvention described herein may be combined to the demonstration of thesurface markers of the cells as previously described for the knowncentromere-specific chromosomal probes [Karenko, L. et al., J. Invest.Dermatol. 116 (2001) 188-193].

The present invention provides a more reliable, earlier and easierdiagnosis of lymphoproliferative diseases, such as CTCL, and opens newpossibilities in the therapy thereof.

The following examples are given for further illustration of theinvention.

EXAMPLE 1

Identification of a Translocation in Chromosome 12

a) Cell Culture and Conventional Chromosome Preparations

Peripheral blood lymphocytes were isolated with Ficoll-Isopaque densitycentrifugation, washed with RPMI 1640 (Gibco BRL, Life Technologies) andcultured for 3 days in RPMI 1640, in the presence of 20% Fetal BovineSerum (Gibco BRL), L-Glutamine (100× Liquid, used as 1×, Gibco BRL),antibiotics (Penicillin 10 000 IU/ml, Streptomycin 10 000 micrograms/ml,used as 1:100), and Phytohemagglutinin (PHA 10576-015, Gibco BRL, usedas 1:100). After this the cells were treated with hypotonicKCl-solution, fixed with glacial acetic acid-methanol (1:3) and the cellsuspension was dropped on objective slides to make conventionalchromosome preparations.

b) MFISH-Method

The air-dried preparations were fixed with 0.1% paraformaldehyde anddried in ethanol series (70%, 85%, 100%). A probe mixture containingpainting probes specific for each chromosome pair labelled with achromosome-specific fluorochrome combination (24XCyte-MetaSystems' 24color kit, MetaSystems GmbH, Altlussheim, Germany) was denatured in a75° C. water bath for 6 minutes, put briefly on ice, and kept 30 to 60minutes in a 37° C. incubator according to the manufacturer'sinstructions. The DNA of the chromosomes was denaturated in 70%formamide in 2×SSC, pH 7.0, for 2 minutes, the slides were dried in 70%,85% and 100% ethanol, respectively, and put on a 37° C. warm plate. Theprobe was applied on the chromosomes on the slides, and a cover slip wassealed with rubber cement (Starkey Chemical CO, IL, USA). The slideswere incubated for 3 to 5 days in a moist chamber at 37° C.

The slides were washed with 50% formamide in 2×SSC, pH 7.3, at 42° C.for 2×5 minutes, then in 2×SSC, pH 7.0 at 42° C. for 2×5 minutes, and4×SSC with 0.01% Tween 20 (=SSCT), pH 7.0, for 1 minute. The biotinlabels were detected with one or two layers of streptavidin-Cy5according to the manufacturer's instructions (B-tect kit, MetaSystemsGmbH), and the preparations were mounted in antifade and DAPI (B-tectkit, Metasystems GmbH).

The metaphases were photographed with UV-microscope (Axioplan imagining2, Zeiss, Germany) and analysed using the computer program Isis ofMetaSystems GmbH with MFISH-program module.

The breakpoints were further defined with a conventional G-banding(Verma, R. S. and Babu, A., Human Chromosomes. Manual of basictechniques, 1st ed. Pergamon Press, New York, 1989), in which it waspossible to find these translocated chromosomes among the markerchromosomes and deleted chromosomes.

1c) SKY-Method (Alternative to MFISH)

The SKY [described by Schröck, E., et al., Science 273 (1996) 494-497]was performed as recommended by ASI (Applied Spectral Imaging). Briefly,a probe mixture (from the SKY kit, ASI) was denatured and incubated at37° C. for one hour. The slides were denatured in 70% formamide/2×SSC,and dehydrated. The probe was applied onto the metaphase spreads, andhybridised at 37° C. for two days. The biotinylated probe was detectedwith avidin-Cy5, and the digoxigenin labelled probe with mouseanti-digoxigenin antibodies followed by a goat anti-mouse antibodyconjugated to Cy5.5.

Either with MFISH or SKY, a breakpoint was observed in the region12q14-12q24 in 5 of 6 patients having Sezary syndrome (SS), theleukaemic form of CTCL, and in 2 patients with the Mycosis fungoides(MF) subtype of CTCL (FIGS. 1 and 2). In 3 patients with SS thechromosome material was translocated to chromosome 18p or 18q12-q21(FIG. 3) and in one patient to chromosome 4 (FIG. 2).

EXAMPLE 2

Characterization of the Translocation

The translocation was further defined with FISH by using locus-specificYAC-probes or BAC-probes (YAC-probes, YAC from CEPH, Fondation JeanDausset, France; BAC-probes from Research Genetics, Groningen, tTheNetherlands). The YACs were grown in AHC broth and purified according tothe instructions of Genomesystems (St. Louis, Mo., USA). The BACs weregrown and purified according the instructions of Research Genetics(Groningen). Both YACs and BACs were labelled with FITC(Fluorescein-12-dUTP, NEN Life Science Products, Inc, Boston, Mass.,USA), Alexa 488® or Alexa 594® (both Molecular probes, Leiden, TheNetherlands) or biotin (Oncor Inc. Gaithersburg, Md., USA) using nicktranslation. The BACs were grown and purified according the instructionsof Research Genetics (Groningen). For the YAC 803-C-2 and BACRP11-359M6, see the chapter “Detailed description of the invention”.

The chromosomes involved were identified with centromere-specific probesof chromosomes 12 and 18, labelled with biotin, (Oncor Inc.Gaithersburg, Md., USA) or with digoxigenin (Oncor Inc). The biotinlabel was detected with two layers of avidin Cy3 (ExtrAvidin-Cy3conjugate, Sigma-Aldrich, Saint Louis, Mo., USA) and biotinylatedanti-avidin antibodies (goat, Vector) between them. The digoxigeninlabel was detected with anti-digoxigenin antibodies made in sheep(Roche, Mannheim, Germany) followed by anti-sheep antibodies made indonkey and labelled with FITC (Jackson West Grove, Pa., USA). Thepreparations were mounted with Vectashield® with DAPI (VectorLaboratories, Inc, Burlingame, Calif., USA).

The metaphases were photographed with UV-microscope (Axioplan imagining2, Zeiss, Germany) and analysed using the computer program Isis ofMetaSystems GmbH with MFISH-program module.

With the method above, the breakpoint was specified to the region12q14-q21.3 in two patients (FIG. 3).

EXAMPLE 3

Identification of NAV3 Gene in the Translocation

For further identification of the translocation additional probes wereused in FISH. YAC 855F7 (CEPH) was grown and purified and labelled withdigoxigenin-11-dUTP (Roche) with nick-translation as described inExample 2. The hybridisation was performed as described above in Example2 above and the hybridised probe was detected with sheepanti-digoxigenin-rhodamine antibodies (Roche, Mannheim).

The hybridised probe was photographed and analysed with ultravioletmicroscope and Isis 3-computer program (FIG. 8). In the sample frompatient 3 suffering from SS, the YAC 855F7 divided between twochromosomes, 12q and 18q. In other hybridisations with further YACs theYACs situated in regions above YAC 855F7 (see FIG. 8, patient responseblock) remained on their respective places in the aberrant 12q, and theYACs below YAC 855F7 were found to be translocated from the aberrant 12qto the aberrant chromosome 18q. In samples from patients 1 and 2, theYAC 855F7 was present only in the normal homologue of chromosome 12, andnot visible in any other chromosome. Only one homologue of the genesrepresented in the YAC855F7, like NAV3, was present in the cells.

YAC 855F7, which is a part of the YAC-contig 12.4 (NIH: www dot ncbi dotnlm dot nih dot gov), spans the region between markers CHLC.GATA65A12and WI-6487. Five consecutive loci, namely LOC255379, LOC255315,LOC204040, LOC121318, and KIAA0938 were found from NCBI database (wwwdot ncbi dot nlm dot nih dot gov) in the corresponding genomic contig(NT_(—)009551). According to PCR of locus and BAC-specific STS-markers(SHGC-155034, G62498, SHGC-79622, and WI-6487, respectively) and BLASTAcomputer analysis (www dot ncbi dot nlm dot nih dot gov), these lociwere found to be situated in YAC 855F7 DNA and in four BACs RP11-781A6,RP11-494K17, RP11-136F16, and RP11-36P3 (accession numbers AC073552.1,AC022268.5, AC073571.14, and AC073608.19, respectively).

The four BACs, RP11-781A6, RP11-494K17, RP11-136F16, and RP11-36P3, werepurified, labelled, hybridised and photographed as described above. Inthe sample from patient 3 suffering from SS, the BAC RP11-781A6 and theBAC RP11-494K17 remained on their places in the aberrant chromosome 12,whereas BAC RP11-136F6 and BAC RP11-36P3 were translocated to chromosome18q. In samples from SS-patients 1 and 2, one homologue corresponding tothe BAC RP11-36P3 was totally absent the signal being present only inthe normal 12q (FIG. 8, enlargement).

According to the BLASTA-analysis and Maes et al (2002), the above loci255315, 204040, 121318 and KIAA0938 together form a RAINB1-genehomologue neuron navigator 3 (NAV3). This gene has Seq. Id. Nr.1(AF397731) or alternatively 3.

The BAC RP11-494K17 contains locus 255315 and almost the whole locus204040. Part of the latter locus is also represented in BAC 136F16, aswell as the whole locus 121318 and a small part of KIAA0938. The BACRP11-36P3 contains the whole locus KIAA0938, but not sequence of locus121318. The BAC 781A6 contains locus 255379 and a small part of locus255315 (FIG. 9). Thus, in the translocation in the sample of patient 3,the NAV3 gene is split into two parts so that the other part remains inchromosome 12q but the other part translocates to chromosome 18q. In thesamples from two other SS-patients studied, at least the lowest part ofthe gene (BAC RP11-36P3) is totally deleted.

1. A method for diagnosis of Sezary syndrome (SS) by detecting thepresence or absence of the neuron navigator 3 (NAV3) gene in a humanpatient, the method comprising: i) analyzing a nucleic acid sample froma human patient to detect the presence or the absence of a neuronnavigator 3 (NAV3) gene having SEQ ID. NO: 1, said absence of the NAV3gene being associated with said SS; ii) identifying a human patient withsaid absence of the NAV3 gene, wherein the absence of the NAV3 gene isindicative of a deletion of the NAV3 gene; and iii) diagnosing SS insaid human patient based on the detection of said absence of the NAV3gene.
 2. A method for diagnosis of Sezary syndrome (SS) by detecting adeletion of the neuron navigator 3 (NAV3) gene in a human patient, themethod comprising: i) a analyzing a nucleic acid sample from a humanpatient to detect the presence or absence of a deletion of a neuronnavigator 3 (NAV) gene having SEQ ID NO: 1, said deletion beingassociated with said SS; ii) identifying a human patient with saiddeletion of the NAV3 gene; and iii) diagnosing SS in said human patientbased on the detection of said deletion of the NAV3 gene.
 3. A method ofclaim 1, wherein the absence of NAV3 gene having SEQ ID. NO: 1 isdetected in chromosome 12 in the region 12q14-12q24.
 4. A method ofclaim 2, wherein the deletion of NAV3 gene having SEQ ID. NO: 1 isdetected in chromosome 12 in the region 12q14-12q24.
 5. A method ofclaim 1, wherein the nucleic acid sample is a metaphase spread.
 6. Amethod of claim 2, wherein the nucleic acid sample is a metaphasespread.